CN105420127A - High-yielding strain of high molecular weight pullulan and method for producing high molecular weight pullulan by utilizing high-yielding strain - Google Patents
High-yielding strain of high molecular weight pullulan and method for producing high molecular weight pullulan by utilizing high-yielding strain Download PDFInfo
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Abstract
The invention relates to a high-yielding strain of high molecular weight pullulan and a method for producing the high molecular weight pullulan by utilizing the high-yielding strain. The high-yielding strain is named as aureobasidium pullulans SWP 35 in a classifying manner, and is preserved in General Microbiology Center of China General Microbiological Culture Collection Center on 11 November, 2015, and the preservation number is CGMCC NO.11602; the method for producing the high molecular weight pullulan by utilizing the high-yielding strain comprises the steps of transferring the slant strain with 4 DEG C in to a PDA culture medium for culturing, inoculating a truffle into a seed culture fluid for culturing to obtain a seed solution, inoculating 1 to 5 percent (v/v) of the seed solution into a fermentation broth, performing fermentation in an airlift type fermentation tank with 40 to 70 percent of liquid volume, performing postprocessing on the fermentation broth to obtain a white pullulan product. The culture medium provided by the invention does not contain an organic nitrogen source, on one hand, the culture medium cost is reduced, on the other hand, pigment in the culture medium is reduced, the alcohol decolorization pressure of pullulan in a postprocessing of fermentation is relieved, the obtained aureobasidium pullulans with the preservation number CGMCC No. 11602 can used for fermenting to produce the pullulan of which Mw is greater than 560000, and meanwhile, the yield reaches to 35g/ or more.
Description
Technical field
The invention belongs to industrial biotechnology field, be specifically related to a kind of superior strain of high molecular pulullan polysaccharide and utilize the method for this bacterial strain production high molecular pulullan polysaccharide.
Background technology
Pulullan polysaccharide (Pullulan), also known as Pul, pullulan, be by Aureobasidium pullulans (
aureobacidiumpullulans) the outer neutral polysaccharide of the water-soluble born of the same parents of a class that secrete.This polysaccharide is the poly-trisaccharide maltose (containing a small amount of maltotetrose) connected with α-1,6-glycosidic link, and generally not having branched structure, is straight-chain polysaccharide.The glycosidic link mode of connection of pulullan polysaccharide uniqueness makes it have the Structural flexibility of height and good water-soluble, thus have that other polysaccharide is unexistent, unique, excellent physical property, comprise good adhesive property, become fibering, film-forming properties and plasticity-.Meanwhile, easily to carry out chemical modification water-soluble or provide active reactive group to change it for pulullan polysaccharide.Therefore, pulullan polysaccharide and derivative thereof are widely used in the various fields such as food, medicine, chemical industry and electronics, are a kind of Multifucntional biological products having very big Development volue and prospect.
At present, the application that pulullan polysaccharide is main is as foodstuff additive, capsule material and cosmetic material.In recent years, because pulullan polysaccharide has the characteristic such as good biocompatibility, polymer itself and the mechanical property that degraded product is all nontoxic, degradation speed is controlled, suitable, excellent formability, the applied research prepared in material, organizational project application and medicine encapsulation macromolecular material at medicine occlusion vehicle, microcapsule obtains a large amount of concern.As polymeric biomaterial, molecular weight and distribution thereof are the important parameters of microbial polysaccharide, affect physical and mechanical properties and the degradation rate of its application product (as tissue engineering bracket etc.).On the other hand, high molecular weight products can significantly improve soltion viscosity.Therefore, the pulullan polysaccharide of production high molecular can expand pulullan polysaccharide Application Areas greatly, improves added value of product.
Fermentable is the main production method of pulullan polysaccharide, and because of the difference of the difference of bacterial classification, medium component, fermentation condition, the molecular weight of pulullan polysaccharide also varies widely.Fermentation method for producing about different molecular weight pulullan polysaccharide also has a lot of report.Patent 97121608.8 discloses a kind of novel method of fermentative production pulullan polysaccharide, and the pulullan polysaccharide molecular-weight average of production is 100,000 dalton; Disclosed in patent 200810052070.6, a kind of fermentation process producing pulullan polysaccharide obtains the pulullan polysaccharide between molecular weight 20-60 ten thousand dalton; Patent 200910148764.4 discloses a kind of method of producing different molecular weight pulullan polysaccharide, can produce the pulullan polysaccharide of molecular-weight average between 80,000-48 ten thousand by regulating organic nitrogen source and inorganic nitrogen-sourced concentration in fermention medium.From the report of these and other published pulullan polysaccharide production method, the molecular weight of the pulullan polysaccharide of production is all lower, all below 600,000 dalton, strongly limit production and the application of pulullan polysaccharide.At present, Fu Ruida bio tech ltd, Shandong (number of patent application: 201210555219.9) by regulating phosphatic concentration and initial pH in substratum is only had in document, obtain the pulullan polysaccharide of molecular weight from 50 ten thousand to 200 ten thousand, but its output for high molecular pulullan polysaccharide has no report, also not for the culture scheme that different molecular weight product design is different.
Because high molecular pullulan fermentation liquid has very high viscosity, the phase shows typical non-newtonian fluid characteristic especially after fermentation, thus has influence on the transmission of the oxygen in fermentation later stage, and this also determines the singularity of whole zymotechnique.At present, the production of pulullan polysaccharide adopts mechanical agitation type fermentor tank more; But there will be serious dissolved oxygen restriction in the high molecular pullulan fermentation later stage, almost remaining static at the outer fermented liquid of stirring rake, in order to improve mixing effect, having to consume a large amount of electric power for cost.
Summary of the invention
The object of this invention is to provide a kind of superior strain of high molecular pulullan polysaccharide and utilize the method for this bacterial strain production high molecular pulullan polysaccharide, high molecular pulullan polysaccharide can be obtained in a large number.
The technical solution adopted in the present invention is:
The superior strain of high molecular pulullan polysaccharide, is characterized in that:
The Classification And Nomenclature of described superior strain be Aureobasidium pullulans (
aureobasidiumpullulans) SWP35, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on November 11st, 2015, deposit number is CGMCCNO.11602.
The superior strain of the high molecular pulullan polysaccharide described in utilization produces the method for pulullan polysaccharide, it is characterized in that:
Comprise the following steps:
(1) actication of culture:
By 4 DEG C of slant preservation bacterial classification Aureobasidium pullulans (
aureobasidiumpullulans) SWP35 is transferred to PDA substratum, cultivate 4 ~ 6 days for 28 ± 2 DEG C;
(2) seed culture:
Choose the flat board of pure culture, use aseptic punch tool and aseptic inoculation pin, bacterium block is inoculated in seed culture fluid, in 28 ± 2 DEG C, under rotating speed 200 ± 30rpm condition, cultivate 24 ~ 48h, obtained seed liquor;
(3) fermentation culture:
By seed liquor obtained for step (2) with 1 ~ 5%(V/V) be inoculated in fermentation culture, ferment in the airlift fermentor of liquid amount 40 ~ 70%, leavening temperature 28 ± 2 DEG C, air flow is 0.5 ~ 1.5VVM, fermentation 60 ~ 96h;
(4) fermented liquid aftertreatment:
, clarification filtration, alcohol precipitation, ethanol wash degerming through Plate Filtration after fermented liquid dilution, press filtration or the centrifugal organic solvent that removes obtain solid substance, can obtain white pulullan polysaccharide product after solid substance drying and pulverizing.
In step (1), the formula of PDA substratum is as follows:
Potato 200g/L, glucose 20g/L, agar 20g/L, surplus is water;
Initial pH nature, 121 DEG C, 20min sterilizing.
In step (2), the formula of seed culture fluid is as follows:
Glucose 50g/L, ammonium sulfate 0.6g/L, yeast powder 1.7g/L, magnesium sulfate 0.2g/L, sodium-chlor 1.0g/L, dipotassium hydrogen phosphate 5.0g/L;
Initial pH6.5,121 DEG C, 20min sterilizing.
In step (3), the formula of fermentation culture is as follows:
Sucrose 50 ~ 100g/L, ammonium sulfate 1.2 ~ 2.1g/L, magnesium sulfate 0.2g/L, sodium-chlor 1.0g/L, dipotassium hydrogen phosphate 5.0g/L, surplus is water;
Initial pH6.5,121 ± 5 DEG C, 20min sterilizing.
The present invention has the following advantages:
The high molecular pulullan yield utilizing technical solution of the present invention to produce is high (being greater than 35g/L), and weight-average molecular weight is greater than 2,000,000, can be used for the field of tissue engineering technology such as hydrogel, artificial bone; Then can significantly reduce pulullan polysaccharide addition for food thickening, reduce use cost.
The present invention adopts airlift fermentor production high molecular pulullan polysaccharide, compares stirred-tank fermenter, consumes energy lower, and the pulullan polysaccharide molecular weight of acquisition is higher; The present invention's fermention medium used is not containing organic nitrogen source, and medium component is clear and definite, and rear extraction process is simple, can obtain high molecular pulullan polysaccharide in a large number, be suitable for suitability for industrialized production.
Accompanying drawing explanation
Fig. 1 is that embodiment one prepares pulullan polysaccharide infrared colour spectrogram.
Fig. 2 is that embodiment two prepares gained pulullan polysaccharide infared spectrum.
Fig. 3 is pulullan polysaccharide standard substance (Sigma) infared spectrum.
Fig. 4 is pulullan polysaccharide molecular weight mark product (Sigma) GPC result.
Fig. 5 is the pulullan polysaccharide molecular mass standard curve obtained by three rank matchings.
Fig. 6 is embodiment a part flow measurement figure.
In Fig. 6, embodiment one sample appearance time is 14.278, by three rank fit equation estimations, and obtained pulullan polysaccharide molecular weight Mw=2359232.
Fig. 7 is embodiment two molecular weight determination figure.
In Fig. 7, embodiment two sample appearance time is 14.067, and by three rank fit equation estimations, the pulullan polysaccharide molecular weight of acquisition is 3330502kDa.
Embodiment
Below in conjunction with embodiment, the present invention will be described in detail.
Experimental technique in following content, if no special instructions, is ordinary method; Experiment material used in following embodiment, if no special instructions, is routine biochemistry reagent.
The present invention utilizes complex mutation technology screening to obtain the superior strain of a plant height molecular weight pulullan polysaccharide, Classification And Nomenclature be Aureobasidium pullulans (
aureobasidiumpullulans) SWP35, from Shaanxi Province of China Luochuan Area apple surface be separated obtain Aureobasidium pullulans (
aureobsidiumpullulans) SW2009AP5 is that starting strain carries out ultraviolet and nitrite complex mutation and obtains, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on November 11st, 2015, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCCNO.11602.
Utilize the superior strain of above-mentioned high molecular pulullan polysaccharide to produce the method for pulullan polysaccharide, comprise the following steps:
(1) actication of culture:
By 4 DEG C of slant preservation bacterial classification Aureobasidium pullulans (
aureobasidiumpullulans) SWP35 is transferred to PDA substratum, cultivate 4 ~ 6 days for 28 ± 2 DEG C;
(2) seed culture:
Choose the flat board of pure culture, use aseptic punch tool and aseptic inoculation pin, bacterium block is inoculated in seed culture fluid, in 28 ± 2 DEG C, under rotating speed 200 ± 30rpm condition, cultivate 24 ~ 48h, obtained seed liquor;
(3) fermentation culture:
By seed liquor obtained for step (2) with 1 ~ 5%(V/V) be inoculated in fermentation culture, ferment in the airlift fermentor of liquid amount 40 ~ 70%, leavening temperature 28 ± 2 DEG C, air flow is 0.5 ~ 1.5VVM, fermentation 60 ~ 96h;
(4) fermented liquid aftertreatment:
, clarification filtration, alcohol precipitation, ethanol wash degerming through Plate Filtration after fermented liquid dilution, press filtration or the centrifugal organic solvent that removes obtain solid substance, can obtain white pulullan polysaccharide product after solid substance drying and pulverizing.
In step (1), the formula of PDA substratum is as follows:
Potato 200g/L, glucose 20g/L, agar 20g/L, surplus is water;
Initial pH nature, 121 DEG C, 20min sterilizing.
In step (2), the formula of seed culture fluid is as follows:
Glucose 50g/L, ammonium sulfate 0.6g/L, yeast powder 1.7g/L, magnesium sulfate 0.2g/L, sodium-chlor 1.0g/L, dipotassium hydrogen phosphate 5.0g/L;
Initial pH6.5,121 DEG C, 20min sterilizing.
In step (3), the formula of fermentation culture is as follows:
Sucrose 50 ~ 100g/L, ammonium sulfate 1.2 ~ 2.1g/L, magnesium sulfate 0.2g/L, sodium-chlor 1.0g/L, dipotassium hydrogen phosphate 5.0g/L, surplus is water;
Initial pH6.5,121 ± 5 DEG C, 20min sterilizing.
The acquisition of high molecular pulullan polysaccharide superior strain:
(1) original strain SW2009AP5 and characteristic thereof: original strain SW2009AP5 be from be separated from Shaanxi Province of China Luochuan Area apple surface obtain Aureobasidium pullulans (
aureobsidiumpullulans).This bacterial strain is when grow on plates, and the bacterium colony initial stage is canescence, after turn dark to brown gradually, in hide-like, have fold, bacterium colony is moistening, and microscopic examination thalline is mycelioid.Adopting containing organic nitrogen source medium liquid culture 96 hours secondary fermentation liquid colors is tawny, and thalline is many, and in yeast shape, exopolysaccharides reaches 23.5g/L, Sucrose conversion 47%, obtains the weight-average molecular weight about 200,000 of pulullan polysaccharide.
(2) acquisition of mutagenic strain: original strain SW2009AP5 obtains mutagenic strain SWP35 through ultraviolet and nitrous acid multiple mutated.
1) preparation of spore suspension: slant strains be inoculated in liquid nutrient medium, 28 DEG C, 230r/min cultivates 48h, reaches logarithmic phase.Put into triangular flask vibration 10 ~ 15min that glass sphere is housed, smash spore agglomerate, filter with absorbent cotton, through the centrifugal 10min of 10000r/min, get stroke-physiological saline solution lotion twice, adopt the method counting of blood counting chamber and dull and stereotyped coating, adjustment spore concentration is to 10
7individual/mL, makes the spore suspension for mutagenic treatment.
2) ultraviolet mutagenesis process: get the above spore suspension 5mL prepared in the sterilizing plate of 9cm.15W ultraviolet lamp opens preheating 20min, is put on magnetic stirring apparatus by the plate filling spore suspension, and distance fluorescent tube 20cm, opens ware lid, irradiate 1 ~ 4min, stir while irradiate.Spore suspension after process carries out different multiples dilution in Bechtop, then spread plate, cultivate 3 days observationss, calculate lethality rate, and it is fast to select those growths, bacterium colony is large, and bacterium colony periphery is that stiff is gluey, the bacterial strain of color desalination carries out preservation, adopts the assessment of carrying out producing pulullan polysaccharide ability containing organic nitrogen source medium.
3) nitrite mutagenic treatment: sift out bacterial strain through ultraviolet mutagenesis process and prepare spore suspension 2mL, add 1mL0.1mol/LNaNO
2solution, then add 1mL1mol/LpH4.5 acetate buffer solution (NaNO
2concentration is 0.025mol/L), 28 DEG C of insulation 30 ~ 120min.After process certain hour, get the Na that 2mL treatment solution adds pH8.6
2hPO
4solution, adjustment pH is 7.0, and mutagenesis stops.Flat board is coated with by after different multiples dilution, cultivate 3 days observationss, calculate lethality rate, and select that growth is fast, bacterium colony large, bacterium colony periphery be that stiff is gluey, the bacterial strain of color desalination carries out preservation, adopt the assessment not carrying out product pulullan polysaccharide ability containing organic nitrogen source medium.
4) mutagenic strain produces pulullan polysaccharide capability evaluation: by 2) or 3) in the bacterial strain of screening be inoculated into respectively in seed culture medium, at 28 DEG C, under 220r/min, shaking culture 48h, with in the inoculum size access fermention medium of 5%, 28 DEG C, 220r/min bottom fermentation 4d, measure the output of pulullan polysaccharide, purity and molecular weight, sift out high molecular pulullan polysaccharide superior strain swp35 again.
5) fermention medium is divided into containing organic nitrogen source medium with not containing organic nitrogen source medium.Containing the product pulullan polysaccharide capability evaluation of organic nitrogen source medium for bacterial strain after ultraviolet mutagenesis process; Not containing the product pulullan polysaccharide capability evaluation of organic nitrogen source medium for bacterial strain after nitrite mutagenic treatment.
Containing organic nitrogen source medium: sucrose 50g/L, ammonium sulfate 0.6g/L, yeast powder 1.7g/L, magnesium sulfate 0.2g/L, sodium-chlor 1.0g/L, dipotassium hydrogen phosphate 5.0g/L;
Not containing organic nitrogen source medium: sucrose 100g/L, ammonium sulfate 0.6g/L, magnesium sulfate 0.2g/L, sodium-chlor 1.0g/L, dipotassium hydrogen phosphate 5.0g/L.
(3) mutagenic strain swp35 characteristic: this bacterial strain is when grow on plates, and colony colour is white, and bacterium colony is rounded, central elevation, smooth surface, and bacterium colony is moistening, and microscopic examination thalline is mycelioid.It is lightpink that employing does not contain organic nitrogen source medium liquid culture 96 hours secondary fermentation liquid colors, and thalline is many, and in yeast shape, exopolysaccharides reaches 32.8g/L, Sucrose conversion 32.8%, and the weight-average molecular weight obtaining pulullan polysaccharide reaches more than 2,000,000.
Embodiment one:
(1) by 4 DEG C of slant preservation bacterial classification Aureobasidium pullulans (
aureobsidiumpullulans) activation of CGMCCNo.11602 use PDA substratum, activation temperature 28 DEG C, soak time 4 days;
(2) by aseptic punch tool and inoculating needle, the bacterium block of pure culture is inoculated in seed culture fluid (filling a prescription the same), 28 DEG C, under rotating speed 200rpm condition, cultivates 48h, obtained seed liquor;
(3) by the seed liquor prepared according to 5%(V/V) amount be inoculated in and be equipped with in the 1L triangular flask of 200mL fermentation culture, wherein medium component is: sucrose 75g/L, ammonium sulfate 1.5g/L, magnesium sulfate 0.2g/L, sodium-chlor 1.0g/L, dipotassium hydrogen phosphate 5.0g/L, initial pH6.5,121 DEG C of sterilizing 20min, rotating speed 180rpm shaking table, 28 DEG C of fermentation culture 96h;
(4) use isopyknic water that fermented liquid is diluted 1 times, stir, rotating speed 6000rpm, the centrifugal 20min of normal temperature, abandons precipitation and stays supernatant liquor, and gained fermented liquid supernatant is carried out diatomite clarification filtration, removes the trickle solid substance do not got rid of in centrifugal process;
(5) in clear filtrate, add 95% industrial spirit precipitation of 1.5 times of volumes, precipitation operation temperature is 25 DEG C, and sedimentation time is 5 hours; Centrifugation, solid substance washs 1 time with a small amount of 95% industrial spirit again, and suction filtration removes organic solvent and obtains solid substance, and solid substance obtains pulullan polysaccharide product through vacuum-drying with after pulverizing;
(6) Aureobasidium pullulans (Aureobsidiumpullulans) CGMCCNo.11602 fermentative production pulullan yield is 30g/L, adopts DNS method to analyze trisaccharide maltose content and show that preparing gained pulullan polysaccharide purity reaches 96.8% after Pullulanase enzymolysis.Infrared chromatography measures and shows, preparing gained pulullan polysaccharide and Sigma, to mark product pulullan polysaccharide infared spectrum consistent; GPC measures and shows, weight-average molecular weight 2,350,000.
Embodiment two:
(1) by 4 DEG C of slant preservation bacterial classification Aureobasidium pullulans (
aureobsidiumpullulans) activation of CGMCCNo.11602 use PDA substratum, activation temperature 28 DEG C, soak time 4 days;
(2) by aseptic punch tool and inoculating needle, the bacterium block of pure culture is inoculated in seed culture fluid (filling a prescription the same), 28 DEG C, under rotating speed 200rpm condition, cultivates 48h, obtained seed liquor;
(3) by the seed liquor prepared according to 5%(V/V) amount be inoculated in and be equipped with in the 10L airlift fermentor of 7L fermentation culture, wherein medium component is: sucrose 100g/L, ammonium sulfate 1.8g/L, magnesium sulfate 0.2g/L, sodium-chlor 1.0g/L, dipotassium hydrogen phosphate 5.0g/L, initial pH6.5,121 DEG C of sterilizing 20min, 28 DEG C, air flow 1VVM, tank pressure 0.5Mpa, fermentation culture 72h;
(4) in storage tank, use isopyknic water that fermented liquid is diluted 1 times, stir, adopt Plate Filtration process to remove thalline, then adopt diatomite filter to carry out clarification filtration to gained fermented liquid supernatant;
(5) in clear filtrate, add 95% industrial spirit precipitation of 1.5 times of volumes, precipitation operation temperature is 20 DEG C, and sedimentation time is 5 hours; Adopt press filtration to make solid-liquid two-phase laminated flow, solid substance uses a small amount of above-mentioned corresponding washing with alcohol 1 time again, and press filtration removes organic solvent and obtains solid substance, and solid substance obtains pulullan polysaccharide product through vacuum-drying with after pulverizing;
(6) Aureobasidium pullulans (Aureobasidiumpullulans) CGMCCNo.11602 fermentative production pulullan yield is 35g/L, adopts DNS method to analyze trisaccharide maltose content and show that preparing gained pulullan polysaccharide purity reaches 97.5% after Pullulanase enzymolysis.Infrared chromatography measures and shows, preparing gained pulullan polysaccharide and Sigma, to mark product pulullan polysaccharide infared spectrum consistent; GPC measures and shows, prepares gained pulullan polysaccharide weight-average molecular weight 3,330,000.
Used medium of the present invention, without organic nitrogen source, reduces culture medium cost on the one hand, reduces pigment in substratum on the one hand in addition, alleviates the pressure of pulullan polysaccharide alcohol decolouring in fermentation aftertreatment.Adopt this substratum, Aureobasidium pullulans CGMCCNo.11602 can the pulullan polysaccharide of fermentative production Mw>2560000, and output reaches more than 35g/L simultaneously.In research in the past, usually be all sacrifice output to exchange high molecular for, thus cause production cost higher, originally the bacterial strain non-pigment licensed, adopt airlift fermentor, reduce the energy consumption of stirring and producing, the pulullan polysaccharide of production is meeting high molecular while, ensure high yield, be suitable as novel super-high Molecularly Imprinted Polymer and carry out industrialization development in fields such as biomaterial, pharmaceutical carrier, beauty treatments.
Content of the present invention is not limited to cited by embodiment, and the conversion of those of ordinary skill in the art by reading specification sheets of the present invention to any equivalence that technical solution of the present invention is taked, is claim of the present invention and contains.
Claims (5)
1. the superior strain of high molecular pulullan polysaccharide, is characterized in that:
The Classification And Nomenclature of described superior strain be Aureobasidium pullulans (
aureobasidiumpullulans) SWP35, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on November 11st, 2015, deposit number is CGMCCNO.11602.
2. utilize the superior strain of the high molecular pulullan polysaccharide described in claim 1 to produce the method for pulullan polysaccharide, it is characterized in that:
Comprise the following steps:
(1) actication of culture:
By 4 DEG C of slant preservation bacterial classification Aureobasidium pullulans (
aureobasidiumpullulans) SWP35 is transferred to PDA substratum, cultivate 4 ~ 6 days for 28 ± 2 DEG C;
(2) seed culture:
Choose the flat board of pure culture, use aseptic punch tool and aseptic inoculation pin, bacterium block is inoculated in seed culture fluid, in 28 ± 2 DEG C, under rotating speed 200 ± 30rpm condition, cultivate 24 ~ 48h, obtained seed liquor;
(3) fermentation culture:
By seed liquor obtained for step (2) with 1 ~ 5%(V/V) be inoculated in fermentation culture, ferment in the airlift fermentor of liquid amount 40 ~ 70%, leavening temperature 28 ± 2 DEG C, air flow is 0.5 ~ 1.5VVM, fermentation 60 ~ 96h;
(4) fermented liquid aftertreatment:
, clarification filtration, alcohol precipitation, ethanol wash degerming through Plate Filtration after fermented liquid dilution, press filtration or the centrifugal organic solvent that removes obtain solid substance, can obtain white pulullan polysaccharide product after solid substance drying and pulverizing.
3. the method utilizing the superior strain of high molecular pulullan polysaccharide to produce pulullan polysaccharide according to claim 2, is characterized in that:
In step (1), the formula of PDA substratum is as follows:
Potato 200g/L, glucose 20g/L, agar 20g/L, surplus is water;
Initial pH nature, 121 DEG C, 20min sterilizing.
4. the method utilizing the superior strain of high molecular pulullan polysaccharide to produce pulullan polysaccharide according to claim 2, is characterized in that:
In step (2), the formula of seed culture fluid is as follows:
Glucose 50g/L, ammonium sulfate 0.6g/L, yeast powder 1.7g/L, magnesium sulfate 0.2g/L, sodium-chlor 1.0g/L, dipotassium hydrogen phosphate 5.0g/L;
Initial pH6.5,121 DEG C, 20min sterilizing.
5. the method utilizing the superior strain of high molecular pulullan polysaccharide to produce pulullan polysaccharide according to claim 2, is characterized in that:
In step (3), the formula of fermentation culture is as follows:
Sucrose 50 ~ 100g/L, ammonium sulfate 1.2 ~ 2.1g/L, magnesium sulfate 0.2g/L, sodium-chlor 1.0g/L, dipotassium hydrogen phosphate 5.0g/L, surplus is water;
Initial pH6.5,121 ± 5 DEG C, 20min sterilizing.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111690547A (en) * | 2020-06-23 | 2020-09-22 | 浙江树人学院（浙江树人大学） | Xylitol strain, fermentation medium, optimization method and application |
| CN112430549A (en) * | 2020-12-11 | 2021-03-02 | 中国海洋大学 | Natural bacterial strain for producing pullulan and application thereof |
| CN113881734A (en) * | 2021-11-16 | 2022-01-04 | 江苏力凡胶囊有限公司 | Culture medium for producing pullulan through microbial fermentation and application of culture medium |
| CN116286366A (en) * | 2023-03-15 | 2023-06-23 | 齐齐哈尔大学 | Microbial separation method for alkaline microbial detection |
| CN116426391A (en) * | 2023-03-28 | 2023-07-14 | 陕西省微生物研究所 | Aureobasidium pullulans Aureobasidium pullulans P1 and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111690547A (en) * | 2020-06-23 | 2020-09-22 | 浙江树人学院（浙江树人大学） | Xylitol strain, fermentation medium, optimization method and application |
| CN112430549A (en) * | 2020-12-11 | 2021-03-02 | 中国海洋大学 | Natural bacterial strain for producing pullulan and application thereof |
| CN113881734A (en) * | 2021-11-16 | 2022-01-04 | 江苏力凡胶囊有限公司 | Culture medium for producing pullulan through microbial fermentation and application of culture medium |
| CN116286366A (en) * | 2023-03-15 | 2023-06-23 | 齐齐哈尔大学 | Microbial separation method for alkaline microbial detection |
| CN116426391A (en) * | 2023-03-28 | 2023-07-14 | 陕西省微生物研究所 | Aureobasidium pullulans Aureobasidium pullulans P1 and application thereof |
| CN116426391B (en) * | 2023-03-28 | 2024-02-23 | 陕西省微生物研究所 | Aureobasidium pullulans Aureobasidium pullulans P1 and application thereof |
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| CN105420127B (en) | 2020-07-03 |
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